Clinicopathological and genomic features of superficial esophageal squamous cell carcinomas in nondrinker, nonsmoker females

Abstract Background Esophageal squamous cell carcinoma (ESCC) is sometimes detected in non‐drinker and non‐smoker females who are considered to have very low risk of ESCC development in daily practice. This study examined the clinicopathological and genomic characteristics of ESCCs in females with no history of drinking and smoking. Methods The sample comprised 118 ESCC lesions occurring in 95 female patients who underwent endoscopic submucosal dissection at our department between January 2008 and December 2019. The patients were categorized into two groups: 51 lesions in 49 patients with no history of drinking and smoking (nondrinker/nonsmoker [NDNS] group) and 69 lesions in 45 patients with a history of drinking or smoking (drinker/smoker [DS] group). We analyzed the differences in clinicopathological and cancerous genomic characteristics between the groups. Significant genomic alterations were validated using immunohistochemistry. Results Multiple logistic regression revealed that older age, fewer multiple Lugol‐voiding lesions (LVLs), and reflux esophagitis (RE) were independently associated with the occurrence of ESCCs in the NDNS group. ESCC lesions in the NDNS group were predominantly located in the mid‐thoracic esophagus, posterior wall side, with 0‐IIa, the aspect ratio of the lesion >2 (vertical/horizontal), and endoscopic keratinization. Genetic analysis showed that CDKN2A driver alterations were significantly more frequent and KMT2D alterations were significantly less frequent in the NDNS group than in the DS group. KMT2D alterations were strongly correlated with immunostaining. Conclusion Older nondrinker, nonsmoker females with RE and fewer multiple LVLs may develop longitudinal 0‐IIa ESCC with keratinization of the posterior wall of the mid‐thoracic esophagus. ESCCs in nondrinker, nonsmoker females had fewer KMT2D alterations and more CDKN2A alterations, which may be a biomarker for treatment.


| INTRODUCTION
4][5] Esophageal carcinomas are of two types: squamous cell carcinoma (SCC) and adenocarcinoma.Epidemiologically, over 80% of esophageal cancers in Asian countries such as Japan are SCC, whereas adenocarcinomas predominate in Western countries. 6lobally, esophageal SCC (ESCC) is more common in males than in females, and in Japan, males are six times more likely to be affected than females. 7,8Consumption of alcohol and tobacco smoking are major risk factors for esophageal cancer, 9,10 and their synergistic effects have also been observed in carcinogenesis. 11They are strongly linked to acetaldehyde metabolism and are mainly regulated by alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2). 12ale sex, alcohol consumption, smoking, ADH1B and ALDH2 gene polymorphisms, and multiple Lugol-voiding lesions (LVLs) significantly affect the incidence of numerous metachronous SCCs. 13,14Therefore, it is crucial to carefully monitor the esophagus in patients with risk factors for ESCC.However, ESCC is sometimes detected in nondrinker and nonsmoker females who are considered to have a very low risk of ESCC development in daily practice.There are few reports on ESCCs in nondrinking and nonsmoking females who have a low risk of ESCC development, and the detailed carcinogenic mechanisms are unclear.
This study aimed to examine the clinicopathological and genomic characteristics of ESCCs in females without a history of smoking and alcohol consumption.

| Patients
We retrospectively enrolled consecutive patients with superficial ESCCs who underwent endoscopic submucosal dissection at Hiroshima University Hospital between January 2008 and December 2019.Males with ESCC were excluded.The included patients were categorized into two groups based on history of drinking or smoking: the nondrinker, nonsmoker group (NDNS group) and the drinker and/or smoker group (DS group) (Figure 1).We analyzed the differences in clinicopathological and genomic characteristics between the groups.

| Definitions
We defined drinkers as patients with a daily drinking habit and smokers as those with a current or past habit of smoking at least one cigarette daily.Esophageal LVLs, reflux esophagitis (RE), degree of gastric atrophy, longitudinal location, macroscopic type, and circumferential location of the esophagus were previously defined [15][16][17] and classified with respect to the Japanese Classification of Esophageal Carcinoma. 18We diagnosed hiatal hernias endoscopically by observing the had fewer KMT2D alterations and more CDKN2A alterations, which may be a biomarker for treatment.

K E Y W O R D S
esophageal neoplasms, esophageal squamous cell carcinoma, genomics, nondrinker, nonsmoker females, reflux esophagitis discordance between the diaphragmatic hiatus and esophagogastric junction. 19A "longitudinal lesion" was defined as a lesion extending along the long axis of the esophageal lumen with a length more than twice that of the short axis.When white keratinizing epithelial adherence was observed in the lesion, it was described as "endoscopic keratinization."

| Pathological examination
We evaluated the specimens according to the Japanese guidelines for esophageal carcinoma. 20The components of intraepithelial carcinoma were classified into two types: the total layer type, in which tumor cells replace the entire epithelial layer, and the basal layer type, in which tumor cells grow mainly in the basal layer and differentiate toward the superficial layer into a more acidophilic cytoplasm.Parakeratosis is characterized by the presence of nuclei in stratum corneum cells, which should not be normally observed.

DNA extraction
We prepared several 10-μmthick slides using formalinfixed paraffin-embedded (FFPE) specimens.From these specimens, we dissected the pathological tumor tissues (cancer) and nontumor tissues surrounding the tumor using the Laser Capture Microdissection System (Leica LMD 6500).We used the GeneRead DNA FFPE Kit (Qiagen, Valencia, CA, USA) to extract DNA from these tissues and the Qubit 1.0 Fluorometer (Life Technologies, Grand Island, NY, USA) to determine DNA concentrations.
Furthermore, we ascertained the quantity and quality of FFPE-derived DNA samples by calculating the normalized DNA integrity scores (ΔΔCq) via quantitative polymerase chain reaction analysis using the Agilent NGS FFPE QC Kit (Agilent Technologies, Santa Clara, CA, USA).

| Target enrichment and next-generation sequencing
We developed a sequencing library based on DNA extracted from the tumors and nontumor mucosa using the SureSelect XT HS Kit (Agilent Technologies) after fragmenting into 150-200 bp using the XT Low Input Enzymatic Fragmentation Kit (Agilent Technologies).The amount of DNA was measured using TapeStation D1000 (Agilent Technologies) before hybridization and used if the prepared library was >500 ng.However, three samples were not obtained.To perform target capture, the SureSelect XT Target Enrichment System (Agilent Technologies) was mounted on 468 cancer genes from the MSK-IMPACT Clinical Sequencing Cohort 22 (Table S1).The resulting pooled libraries were sequenced by paired-end reads using the HiSeq X platform (Illumina, San Diego, CA, USA) after the quality control check with the High Sensitivity D1000 ScreenTape Assay (Agilent Technologies) (Figure 2).

| Variant detection
Sequencing reads were preprocessed using fastp v0.20 and mapped to hg19 using BWA-MEM v0.7.17. 23GATK best practice was used for variant calling.To reduce

| Immunohistochemistry
Sections of paraffin-embedded human esophageal cancer tissue specimens (2-3-μmthick) were mounted on positively charged slides.Antigen retrieval was performed in Tris-EDTA buffer (pH 6.0) in a microwave oven at 800 W for 30 min.Subsequently, the tissues were incubated with the primary antibodies p16 and KMT2D at room temperature (20°C-25°C).Bound primary antibodies were detected using the Dako EnVision System (Dako, Copenhagen, Denmark).The slides were then counterstained using hematoxylin after immunostaining.The immunohistochemical evaluations were performed blinded with regard to the histological diagnosis.

| Statistical analyses
Categorical variables were compared using the Chi-square and Fisher's exact tests.Continuous variables were compared using the Student's t-test.Multivariate logistic regression analysis was performed with stepwise selection.
The Kappa statistic was used to determine the correlation between genomic alterations and KMT2D and CDKN2A immunohistochemistry.All statistical analyses were performed using JMP version 15 (SAS Institute Inc., Cary, North Carolina, USA).p < 0.05 was considered statistically significant.

| Patient characteristics
There were 48 and 47 patients in the NDNS and DS groups, respectively.The characteristics of the patients are presented in Table 1

| Tumor characteristics
There were 50 and 68 lesions in the NDNS and DS groups, respectively.The characteristics of the tumor are presented in Table 3.The mean tumor size and horizontal location ≤1/4 were not significantly different between the groups; however, the mid-thoracic esophagus in the longitudinal location (72.0%[36/50]

| Immunohistochemistry
The immunohistochemistry results for KMT2D and p16 are shown in  7).Regarding the correlation between alterations and immunostaining, the kappa statistic was 0.8924 and 0.1524 for KMT2D and CDKN2A, respectively (Figure 7).
Three of the six cases with KMT2D mutations and low VAF (<0.1) were heterogeneous in immunohistochemistry.

| DISCUSSION
Our study revealed that older nondrinker, nonsmoker females with RE and fewer multiple LVLs might develop longitudinal 0-IIa ESCC with keratinization of the posterior wall of the mid-thoracic esophagus.These lesions had a low frequency of KMT2D alterations and a high frequency of CDKN2A alterations, with less KMT2Dpositive and more p16-positive immunostaining.KMT2D alterations were strongly correlated with immunostaining findings.Multiple LVLs, which are useful predictors of the risk of metachronous multiple ESCCs, are caused by direct exposure to carcinogens, including alcohol consumption and smoking.Therefore, in females who do not drink or smoke, ESCCs may develop from the background mucosa with a very low risk of ESCC without multiple LVLs, implying that the carcinogenic pathways differ.Chronic RE is typically the predominant causative factor for developing Barrett's esophagus and its progression to esophageal adenocarcinoma. 6However, some reports have demonstrated a role for gastroesophageal reflux disease in laryngopharyngeal carcinogenesis and identified a relationship between RE and ESCC by reviewing surgical cases of esophageal cancer. 26,27In our study, multivariate analysis revealed that RE was a risk factor for ESCC development in nondrinking, nonsmoking females.In many symptomatic cases, proton pump inhibitors or potassium-competitive acid blockers, medications, and Barrett's esophagus also reflected the higher incidence of RE in the NDNS group.Shigaki et al. reported that nondrinking, nonsmoking esophageal cancer was more common in older women. 280][31] Estrogen suppresses RE by enhancing the defense against damage to the esophageal mucosa. 32herefore, it is possible that RE increased in older females with a decline in estrogen levels after menopause and may have contributed to the development of ESCC.In studies of ESCC in nondrinking, nonsmoking women, only this study, which had a larger number of cases than previous reports, 28,33,34 found significant differences in all clinical findings of older age, RE, and fewer multiple LVLs.
In the current study, the characteristic endoscopic finding of ESCC in nondrinking, nonsmoking women was longitudinal morphology with keratinization of the posterior wall of the mid-thoracic esophagus.The posterior wall of the mid-thoracic esophagus is where the refluxed stomach acid and contents are retained in the supine position. 35Therefore, based on morphology and location, it is speculated that retention of refluxed gastric acid and stomach contents in the posterior wall of the mid-thoracic F I G U R E 3 Alteration profiling of 32 lesions in the NDNS group and 37 in the DS group.The main panels contain the mutation patterns of 40 genes with more than 4% somatic mutations from 32 and 37 lesions in the NDNS and DS groups, respectively.Pink, yellow, orange, green, beige, blue, red, black, and gray cells indicate nonsense mutations, splice site mutations, frameshift mutations, missense mutations, in-frame indels, deletions, amplifications, multiple hits, and complex events, respectively.The bar graph on the right shows the frequency of mutations in each gene.The horizontal bar indicates whether the cases were in the DS (red) or NDNS (blue) group.DS, drinker and/or smoker; NDNS, nondrinker nonsmoker.esophagus led to the occurrence of ESCC in nondrinking, nonsmoking women.Notably, older women with increasing kyphosis due to osteoporosis can develop hiatal hernia of the esophagus because of decreased abdominal cavity volume, and severe and refractory RE is likely to develop due to reflux of gastric contents. 361][42] There were more cases of 0-IIa ESCC with endoscopic keratinization in the NDNS group.A previous study reported that ESCC in nondrinking, nonsmoking females showed more endoscopic keratinization and more 0-IIa cases and that endoscopic keratinization corresponded to histopathologic parakeratosis. 34,43ndoscopic keratosis, in this case, may have developed through a similar process of repair and regeneration, resulting in changes including mucosal thickening and keratinization in esophagitis related to esophageal achalasia. 37hese findings suggest that the mechanism of longitudinal ESCC with endoscopic keratinization in the posterior Comparison of genetic alterations between the NDNS and DS groups.Red, gray, blue, orange, green, black, pink, and yellow cells indicate amplification, complex event, deletion, frameshift mutation, missense mutation, multi-hit, nonsense mutation, and splice site mutation, respectively.DS, drinker and/or smoker; NDNS, nondrinker nonsmoker.wall of the mid-thoracic esophagus is due to persistent esophageal reflux and retention of refluxed gastric acid, duodenal fluid, and food, leading to chronic hyperplastic esophagitis and eventually to malignant transformation of esophageal epithelial cells via a dysplasia-carcinoma sequence.

NDNS group
Due to recent advances in next-generation sequencing (NGS) technology, which has facilitated analysis of the whole genome, exome, and target sequences, a great deal of information on the aberrations of cancer-related genes causing the development and progression of ESCC has accumulated, leading to the description of the Note: We compared the percentage of deletions and mutations in KMT2D and CDKN2A alterations between the two groups.There were 32 lesions in the NDNS group and 37 lesions in the DS group.

T A B L E 6
Outcome of immunohistochemistry analysis of ESCCs in females.][46] Moreover, in our previous study, we performed NGS focusing on early esophageal cancer and reported that somatic alterations such as TP53, NOTCH1, and CDKN2A deletion are more frequent in cancerous mucosa than in noncancerous mucosa. 47The PI3K/AKT signaling pathway plays an important role in the development of a variety of human carcinomas; PIK3CA mutations in studies of ESCC have been detected in 2.2%-11.8% of analyzed cases.NFE2L2 is a main transcription regulator of the stress response, and its mutations have been detected in 5.0%-11.4% of patients with ESCC.In this study, TP53, NOTCH1, PIK3CA, and NFE2L2 were among the most common genetic mutations, but there was no difference between the NDNS and DS groups.However, KMT2D alterations were less frequent, and CDKN2A alterations were more frequent in the NDNS group than in the DS group.Frequent mutations in the genes involved in histone modifications, including KMT2D, have been found in ESCC.KMT2D mutations occur in 18% of ESCC cases, most of which result in truncated proteins lacking the key methyltransferase domain, indicating a tumor suppressor role for KMT2D in ESCC. 48,49Our results showed that the DS group had a significantly higher frequency of KMT2D alterations and 70% more nonsense mutations than the NDNS group.Mutation location also showed inactivation of all DNA domains after 2409.A study of smoking-related tumors found that KMT2D alterations were predominantly more common in patients with a smoking history and were considered smokingrelated genetic mutations. 50In our study, nearly all 15 patients with KMT2D alterations in the DS group had a smoking history, whereas none of the patients in the NDNS group without a smoking history had KMT2D alterations.Among ESCCs, this is the only study to report that KMT2D alterations are more common in patients who smoke.Therefore, KMT2D was rarely found in the NDNS group without a smoking history, suggesting that it is a crucial genetic variant in the DS group, particularly in cases with a smoking history.CDKN2A is a gene involved in the cell cycle regulatory pathway that is mutated in 8% of ESCCs and is the driver gene for ESCC. 51CDKN2A encodes the cyclin-dependent kinase inhibitor p16 (Ink4a).p16INK4A is a well-known surrogate marker for human papillomavirus (HPV) infection. 52In smokers, HPV infection increases the risk of ESCC development. 53In our study, CDKN2A alterations were more common in the NDNS group than in the DS group.No significant difference was observed in the frequency of CDKN2A deletions; however, a significant difference was found in the frequency of mutations.The location of the mutation in the NDNS group also suggests the inactivation of Ank_2 in the DNA-binding domain.Similar to the results of this study, a study of oral floor cancer found no significant difference in the frequency of deletions in the nondrinking, nonsmoking group compared with the drinking and smoking groups, with predominantly more mutations found. 54nozato et al. also reported that CDKN2A gene variants were significantly more abundant in the background epithelium of patients with ESCC without risk factors, including alcohol consumption and smoking. 55This is the first genetic analysis of ESCC in nondrinking, nonsmoking women, where CDKN2A mutations were more common in tumor areas than in nontumor areas.Additionally, p16 positivity, an indirect marker of HPV infection, was more common in the NDNS group, suggesting more HPV-positive cases among nondrinking, nonsmoking females.The correlation between CDKN2A alterations and p16 was not completely consistent, and many cases were CDKN2A-negative and p16-positive.CDKN2A alterations in the presence or absence of HPV infection in head and neck cancer have been reported, with alterations being more common in HPV-negative cases, 56 indicating that CDKN2A alterations and p16 are not completely consistent.Therefore, there is a distinctive carcinogenic pathway in ESCC in the NDNS group that is not associated with known risk factors, suggesting that CDKN2A plays a vital role in this pathway.This study has some limitations.First, it was a singlecenter, retrospective study.Therefore, a large-scale, multicenter prospective study should be conducted to confirm our findings.Second, 24-h multichannel intraluminal impedance-pH (MII-pH) monitoring should be conducted to verify that RE was an essential factor in ESCC development in the NDNS group.Third, genetic analysis was challenging to perform in all cases.We attempted to extract DNA by LMD in all cases.However, due to the condition of the specimens and their small diameter, only 69 lesions could be evaluated.Therefore, further genetic analysis is needed to elucidate the origin of the NDNS group tumors.Fourth, we observed cases with low VAF that were relatively homogeneous in immunohistochemistry.The reason for this may be that these were epithelium and lamina propria mucosa cases, and normal tissue may have been included during laser microdissection.This could be a limitation of the laser microdissection technique.Fifth, we observed positive immunostaining in nonsense mutations in KMT2D.One possibility is that the nonsense mutation is located after 2409 with the DNA domains up to 2409 remaining functional, causing positive immunostaining.Functional analysis was needed for accurate evaluation but was unavailable in our study.We consider this a limitation.Sixth, immunostaining for p16 suggested that HPV infection was more common in the NDNS group.We could not determine whether HPV infection was related to carcinogenesis of esophageal cancer in nondrinking, nonsmoking females because we did not perform functional analysis.We plan to investigate this in a future study.Seventh, the findings of a CDKN2A copy number of 0 on NGS analysis were not verified using microarray or realtime polymerase chain reaction, and complete deletion of the copy number could not be confirmed.

NDNS
In conclusion, older nondrinker, nonsmoker females with RE and few multiple LVLs may develop longitudinal 0-IIa ESCC with keratinization of the posterior wall of the mid-thoracic esophagus and have fewer KMT2D alterations and more CDKN2A alterations.However, even in nondrinkers and nonsmokers at low risk of ESCC development, caution should be exercised, and endoscopy should be performed in older patients with RE and fewer multiple LVLs.Additionally, CDKN2A mutations are common in ESCC in nondrinker, nonsmoker females, which may have implications for drug discovery and selection.

F I G U R E 5
Distribution of KMT2D and CDKN2A somatic mutations.This shows the location of mutated gene positions for ESCCs in the NDNS and DS groups on the KMT2D and CDKN2A structures.DS, drinker and/or smoker; ESCC, esophageal squamous cell carcinoma; NDNS, nondrinker, nonsmoker.T A B L E 5 Breakdown of KMT2D and CDKN2A alterations in genetic analysis of ESCC in females.

F I G U R E 6
Immunostaining of specimens treated by ESD.Immunostaining of a case negative for KMT2D (A) and p16 (B) immunostaining is shown on the left, while that of a case positive for KMT2D (C) and p16 (D) immunostaining is shown on the right.ESD, endoscopic submucosal dissection.

Table 6
We performed multivariate analysis to predict risk factors for ESCC in the NDNS group after selecting factors using the stepwise method.
and Figure6.Immunohistochemistry for KMT2D and p16 was performed on 50 and 68 lesions Note: We divided all 95 patients into two groups: NDNS group (n = 48) and DS group (n = 47).Abbreviations: DS, drinker and/or smoker; ESCC, esophageal squamous cell carcinoma; ESD, endoscopic submucosal dissection; LA, Los Angeles; NDNS, nondrinker nonsmoker; PPI, proton pump inhibitor; P-CAB, potassium-competitive acid blocker; SD, standard deviation.aThereareduplicated cases.T A B L E 1 Characteristics of female patients who underwent ESD for ESCC.T A B L E 2 Multivariate analysis of predictors of ESCC in nondrinking, nonsmoking females.Note: in the DS group.CDKN2A alterations and positive immunostaining for p16 were found in 87.5% (7/8) and 50% (1/2) of patients in the NDNS and DS groups, respectively (Figure